Project summary/ abstract In 2009, my laboratory discovered the enzymatic activities of the three mammalian TET (Ten-Eleven-Translo- cation) proteins, TET1, TET2 and TET3. Since then, work from our own and other groups has implicated TET proteins in regulating gene expression, cell lineage specification, embryonic development, neuronal function and cancer. TET proteins are dioxygenases that oxidize 5-methylcytosine (5mc) to 5-hydroxymethylcytosine (5hmC) and further oxidized methylcytosines (oxi-mC). They have two biochemical functions: to generate oxi- mC and to facilitate DNA demethylation. However, the mechanisms by which TET proteins exert their diverse biological effects are much less understood. TET2 mutations are frequently observed in myeloid malignancies, but many other cancers are documented to have low 5hmC levels, implying profound loss of TET function even in the absence of TET coding region muta- tions. Because TET loss-of-function is associated with increased DNA methylation, the tumor suppressive role of TET proteins has been assumed to involve their ability to maintain DNA in a demethylated state. However, in powerful, inducible mouse models developed in our laboratory, we have shown that acute deletion of both Tet2 and Tet3 rapidly induces an aggressive, transmissible myeloid leukemia within 4 weeks and with 100% penetrance. Using this system, we have found that while the average level of DNA methylation increases across expressed genes in early hematopoietic stem/precursor cells as expected, there is little or no correla- tion of increased or decreased DNA methylation with up- or down-regulation of gene expression or with onco- genesis. Instead, we observe a strong correlation of oncogenic transformation with increased phospho-H2AX and impaired DNA damage repair. Here we propose to extend these studies to address the mechanisms involved. In this project we will use mouse models as well as in vitro systems to analyze the mechanisms of oncogenesis induced by TET loss-of-function. We will examine the role of TET catalytic activity, and compare the conse- quences of loss-of-function of TET proteins versus DNA methyltransferases (DNMTs). We will examine the kinetic relation between loss of oxi-mC in expanding cells and the development of replication stress, genome instability and chromosomal aberrations. As feasible, we will perform RNAi/CRISPR screens to identify important players that regulate cell expansion induced by TET loss-of-function. We will extend our findings to human cancers with high and low 5hmC. Our studies have the potential to change current paradigms and suggest new therapeutic approaches, by defining the mechanisms by which TET function is linked to genome stability.